Nitrobenzene

To study the damage of various antibiotics treatments on the bacterial cell membrane, E. coli and K. pneumoniae (OD₆₀₀ = 0.5) were inoculated into LB broth containing quercetin (½ MIC), colistin (½ MIC), and quercetin (½ MIC)+colistin (½ MIC) (Qu et al., 2019 (link)), followed by incubation in a shaker (180 rpm) at 37°C for 24 h. Then, these suspensions were centrifuged at 5,000 rpm for 5 min. The supernatant was collected and subjected to the alkaline phosphatase (ALP) activity test via a corresponding kit (Solarbio, Beijing). All experiments were performed in triplicate. In general, AKP/ALP uses p-nitrophenyl phosphate (pNPP) as a phosphatase substrate which turns yellow (λ_max = 405 nm) when dephosphorylated by ALP.
Similarly, the β-galactosidase activity in the supernatant was tested via its corresponding kit (Solarbio, Beijing). β-galactosidase decomposes p-nitrobenzene-β-d-galactopyranoside into p-nitrophenol, and the activity of β-galactosidase is calculated by measuring its absorbance at 420 nm.

AnimalsAdult male western albino rats weighing 150-200 g purchased from the animal house of science college, King Saud University, Riyadh were used throughout this study. The animals were fed on standard pellet diet and water ad libitum. The animals were maintained in a controlled environ-ment under standard conditions of temperature and humidity with an alternating light- and– dark cycle. All the procedures described were reviewed and approved by the King Saud University Animal Ethics Committee.
Dosage and treatmentRats were randomly divided into two groups with ten rats in each. The first group served as a control. On the eighth day, the animals of the second group given an oral dosage of PA at the dose of (250 mg/kg body weight/day for three days; n= ten) (24 (link)). On the third day of PA administration, the rats were scarified and the liver organ was isolated.
Sample preparationBlood collection for estimation of AST, ALT and ALP The blood was collected from retro-orbital plexus without the use of anticoagulant. The blood was allowed to stand for 10 min before being centrifuged at 2000 rpm for 10 min to obtain serum for analysis of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP)
Tissue preparationLiver tissue was washed in ice cold normal saline and homogenized in a homogenizing buffer (50 mM Tris- HCl, 1.15% KCl pH 7.4) using Teflon homogenizer. The homogenate was centrifuged at 4000 g for 20 minutes to remove debris and kept at−80 °C until further use. The supernatant was used for the estimation of glutathione, lipid oxidation, glutathione-S-transferase, lactate dehydrogenase, potassium, sodium and vitamin C.
Biochemical analysisSerum alanine aminotransferase (ALT)ALT was estimated by the method of Reitman and Frankel (25 (link)). Briefly, 0.5 ml of substrate (2 mM α-ketoglutarate, 0.2 M DL-alanine in 0.1M phosphate buffer pH 7.4) was incubated at 37 ^oC for 5 minutes. 0.1 ml of freshly prepared serum was added to the aliquot and again incubated at 37 ^oC for 30 minutes. At the end of incubation, 0.5 ml of 2, 4-dinitrophenyl hydrazine was added, and the aliquot left for 30 minutes at room temperature. 0.5 ml of 0.4 N NaOH was added, and the aliquot was again left for 30 minutes. Absorbance was then recorded at 505 nm against water blank.
Serum aspartate aminotransferase (AST)AST was estimated by the method of Reitman and Frankel (25 (link)). The substrate, however, was 2 mM α-ketoglutarate, 0.2 M DL-aspartate, and the rest of the procedure was similar to ALT measurement method.Serum alkaline phosphatase (ALP) Serum alkaline phosphatase activity was measured according to the method of King and Armstrong (26 (link)), using disodium phenyl phosphate as substrate. The colour developed was read at 510 nm.
Measurement of lipid peroxidationLipid oxidation was evaluated by measuring the levels of lipid peroxidation by-products as thiobarbituric acid reactive substances (TBARS), namely malondialdehyde (MD), using the method of Ruiz-Larrea et al. (27 (link)). Accordingly, the samples were heated with TBA at low pH and the formation of a pink chromogen was measured by absorbance at 532 nm. The concentration of lipid peroxides was calculated as μ moles/ml using the extinction coefficient of MD.
Assay of vitamin CAssay of vitamin C was performed according to the method of Jagota and Dani (28 (link)). 0.2 ml of liver homogenate was mixed with 0.8 ml of 10% trichloroacetic acid (TCA) and incubated in ice for 5 minutes. The samples were then centrifuged for 10 minutes at 3500 rpm and 4°C. 1.5 ml double distilled water was subsequently added to 0.5 ml of the supernatant. Eventually, 2 ml of Folin-phenol reagent was added and absorbance was measured at 760 nm after 10 minutes.
Assay of glutathione (GSH)GSH content was determined according to the method described by Beutler et al. (29 (link)) using 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) with sulfhy-dryl compounds to produce a relatively stable yellow color.
Glutathione-S-transferase (GST) assayThe GST activity was determined by the method of Habig et al. (30 (link)). The 1-chloro-2-4-di-nitrobenzene (CDNB) is neutralized by the enzyme in the presence of GSH as a cosubstrate. The change in absorbance is measured at 340 nm and the activity is expressed as nmol/min/mg protein.
Assay of lactate dehydrogenase (LDH)The quantitative determination of LDH in the brain homogenates was performed using the lactate-to-pyruvate kinetic method described by Henry et al. (31 ).
Determination of potassium levelsPotassium levels were measured in a protein-
free alkaline medium by reaction with sodium tetraphenyl boron, which produced a colloidal suspension. The turbidity of such a suspension is proportional to the potassium concentrations in the range of 2–7 mmol/ l (32 (link)).
Determination of sodium levels Sodium levels were assayed by enzymatic determination of sodium, i.e., the measurement of sodium-dependent galactosidase activity using ortho-Nitrophenyl- β- galactoside (ONPG) as a substrate (33 )
Statistical analysisThe values are expressed as mean ± standard error of the mean (SEM). The results were evaluated using the SPSS (version 12.0) and Origin 6 softwares and evaluated by One -way ANOVA complemented with the Dunnett’s test for multiple comparisons. Statistical significance was considered when The p-value was < 0.05

Nitrobenzene (≥99%
Sigma-Aldrich), cyclohexane (≥99% Sigma-Aldrich), acetonitrile
(≥99.8% Fisher Scientific), and methanol (≥99.8% Fisher
Scientific) were purchased and used without further purification.
UV–visible absorption spectra of nitrobenzene in deionized
water, methanol, acetonitrile, and cyclohexane were recorded using
a SHIMADZU UV-3600i Plus spectrophotometer.
Femtosecond TAS
experiments have been described previously.^{64 (link)} Briefly, femtosecond laser pulses were generated from a regenerative
amplifier seeded by a Ti:sapphire oscillator (Coherent Astrella-HE-USP).
For UV excitation, tunable laser pulses were generated using an optical
parametric amplifier (OPA; Coherent OPerA Solo). Transient absorption
spectra were recorded using a commercial transient absorption spectrometer
(Ultrafast Systems HELIOS Fire) following photoexcitation at 355 nm
(3.49 eV) with pulse energies of 500 nJ at the sample. The broadband
probe beam was created via white-light generation by focusing a small
portion of the 800 nm fundamental beam into a calcium fluoride plate
to give a probing range of 350–650 nm. The relative polarizations
of the pump and probe beam were set at the magic angle of 54.7°.
Nitrobenzene solutions were flowed continuously at 10 mL min^–1 through a Harrick flow cell using a liquid diaphragm pump (KNF,
SIMDOS 02). The concentrations and path lengths were selected to give
an absorbance of around 0.3 at 355 nm: 15 mM water (1000 μm),
104 mM cyclohexane (250 μm), 68 mM acetonitrile (250 μm),
and 102 mM methanol (250 μm). NMR studies indicate negligible
aggregation in 15 mM aqueous nitrobenzene. The instrument response
functions were determined by fitting them to solvent-only spectra.

For the microsolvation
calculations, nitrobenzene was optimized on the S₀ surface
using the CAM-B3LYP^{70 (link)}/Def2-TZVP^{71 (link)} method
employing empirical dispersion with Becke–Johnson damping (GD3BJ).^{72 (link)} The analysis of the vibrational frequencies
confirmed this geometry as a minimum. Molecules of each solvent system
were positioned by hand around the nitrobenzene molecule to maximize
interactions with the NO₂ group, and the resulting microsolvated
system was reoptimized at the CAM-B3LYP/Def2-SVP level of theory.
The analysis of the vibrational frequencies again confirmed the resulting
microsolvated systems as minima. These calculations were performed
using the Gaussian 16 program.⁷³ Following our earlier work,^{29 (link)} all excited-state
calculations on the various gas-phase nitrobenzene structures were
performed at the CASPT2 level, with the same CAS(14,11) active space
as before, which includes the π-orbitals on the ring and the
lone pairs on the nitro-oxygen atoms.^{29 (link)} The basis set was the atomic natural orbital (ANO) of S-type double-ζ
with polarization (ANO-S-VDZP).^{74 (link)} The geometries
were taken from our earlier work^{29 (link)} and
calculations were performed using the OpenMolcas program 2023.^{75 (link)} The CASSCF wave functions were state averaged
over 6 singlets and 5 triplets, except for the triplet excitations
from the T₁(n_Aπ*) and T₂(π_Oπ*) minima which included 7 triplet states. Solvation
effects were included using a water PCM, with the equilibrium charges
from the ground state used for nonequilibrium PCM calculations of
the excited states. Structure optimizations were all at the CASSCF
level.